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Patterns of gene expression, cell growth and cell-type specification during development are often regulated by morphogens. Morphogens are signalling molecules produced by groups of source cells located tens to hundreds of micrometers distant from the responding tissue and are thought to regulate the fate of receiving cells in a direct, concentration-dependent manner. The mechanisms that underlie scalable yet robust morphogen spread to form the activity gradient, however, are not well understood and are currently intensely debated. Here, based on two recent publications, we review two in vivo derived concepts of regulated gradient formation of the morphogen Hedgehog (Hh). In the first concept, Hh disperses on the apical side of developing epithelial surfaces using the same mechanistic adaptations of molecular transport that DNA-binding proteins in the nucleus use. In the second concept, Hh is actively conveyed to target cells via long filopodial extensions, called cytonemes. Both concepts require the expression of a family of sugar-modified proteins in the gradient field called heparan sulphate proteoglycans as a prerequisite for Hh dispersal, yet propose different — direct versus indirect — roles of these essential extracellular modulators.

Hedgehog (Hh) morphogens are involved in embryonic development and stem cell biology and, if misregulated, can contribute to cancer. One important post-translational modification with profound impact on Hh biofunction is its C-terminal cholesteroylation during biosynthesis. The current hypothesis is that the cholesterol moiety is a decisive factor in Hh association with the outer plasma membrane leaflet of producing cells, cell-surface Hh multimerization, and its transport and signaling. Yet, it is not decided whether the cholesterol moiety is directly involved in all of these processes, because their functional interdependency raises the alternative possibility that the cholesterol initiates early processes directly and that these processes can then steer later stages of Hh signaling independent of the lipid. We generated variants of the C-terminal Hh peptide and observed that these cholesteroylated peptides variably impaired several post-translational processes in producing cells and Hh biofunction inDrosophila melanogastereye and wing development. We also found that substantial Hh amounts separated from cholesteroylated peptide tagsin vitroandin vivoand that tagged and untagged Hh variants lacking their C-cholesterol moieties remained bioactive. Our approach thus confirms that Hh cholesteroylation is essential during the early steps of Hh production and maturation but also suggests that it is dispensable for Hh signal reception at receiving cells.

The development of tissues and organs requires close interaction of cells. To achieve this, cells express adhesion proteins such as the neural cell adhesion molecule (NCAM) or its

The development of tissues and organs requires close interaction of cells. To do so, cells express adhesion proteins such as the neural cell adhesion molecule (NCAM) or its Drosophila orthologue Fasciclin 2 (Fas2). Both are members of the Ig-domain superfamily of proteins that mediate homophilic adhesion. These proteins are expressed as different isoforms differing in their membrane anchorage and their cytoplasmic domains. To study the function of single isoforms we have conducted a comprehensive genetic analysis of fas2. We reveal the expression pattern of all major Fas2 isoforms, two of which are GPI-anchored. The remaining five isoforms carry transmembrane domains with variable cytoplasmic tails. We generated fas2 mutants expressing only single isoforms. In contrast to the null mutation which causes embryonic lethality, these mutants are viable, indicating redundancy among the different isoforms. Cell type specific rescue experiments showed that glial secreted Fas2 can rescue the fas2 mutant phenotype to viability. This demonstrates cytoplasmic Fas2 domains have no apparent essential functions and indicate that Fas2 has function(s) other than homophilic adhesion. In conclusion, our data propose novel mechanistic aspects of a long studied adhesion protein.

Metazoan Hedgehog (Hh) morphogens are essential regulators of growth and patterning at significant distances from their source, despite being produced as N-terminally palmitoylated and C-terminally cholesteroylated proteins, which firmly tethers them to the outer plasma membrane leaflet of producing cells and limits their spread. One mechanism to overcome this limitation is proteolytic processing of both lipidated terminal peptides, called shedding, but molecular target site requirements for effective Hh shedding remained undefined. In this work, by using Drosophila melanogaster as a model, we show that mutagenesis of the N-terminal Cardin–Weintraub (CW) motif inactivates recombinant Hh proteins to variable degrees and, if overexpressed in the same compartment, converts them into suppressors of endogenous Hh function. In vivo, additional removal of N-palmitate membrane anchors largely restored endogenous Hh function, supporting the hypothesis that proteolytic CW processing controls Hh solubilization. Importantly, we also observed that CW repositioning impairs anterior/posterior compartmental boundary maintenance in the third instar wing disc. This demonstrates that Hh shedding not only controls the differentiation of anterior cells, but also maintains the sharp physical segregation between these receiving cells and posterior Hh-producing cells.

Cell fate determination during development often requires morphogen transport from producing to distant responding cells. Hedgehog (Hh) morphogens present a challenge to this concept, as all Hhs are synthesized as terminally lipidated molecules that form insoluble clusters at the surface of producing cells. While several proposed Hh transport modes tie directly into these unusual properties, the crucial step of Hh relay from producing cells to receptors on remote responding cells remains unresolved. Using wing development in Drosophila melanogaster as a model, we show that Hh relay and direct patterning of the 3–4 intervein region strictly depend on proteolytic removal of lipidated N-terminal membrane anchors. Site-directed modification of the N-terminal Hh processing site selectively eliminated the entire 3–4 intervein region, and additional targeted removal of N-palmitate restored its formation. Hence, palmitoylated membrane anchors restrict morphogen spread until site-specific processing switches membrane-bound Hh into bioactive forms with specific patterning functions., eLife digest Each cell in a developing embryo receives information that determines what type of body structure it will form. In fruit flies, this information is partly given by a protein called Hedgehog. In the embryo cells that receive it, Hedgehog can trigger a series of events which activate certain genes and thereby regulate structure formation. The Hedgehog proteins are produced by a different organizing group of cells: from there they transport within the embryo, creating a gradient. Depending on where a responding cell is in the embryo, it receives a different amount of Hedgehog, which gives the cell its identity. For example, Hedgehog proteins form a gradient across a fruit fly’s developing wing, which creates a visible vein pattern. How Hedgehog proteins form gradients is enigmatic, however, because once produced, they cling to the cells that created them. The reason for this unusual behavior is that the two ends of the Hedgehog protein are attached to a different fat molecule. In particular, one extremity is linked to a fat molecule called palmitate. These ends’ fatty additions anchor Hedgehog to the cells that produced them. Then, the tethered proteins gather together to form chain-like clusters where they inactivate each other: the extremity with the palmitate ‘hides’ the portion of the neighboring protein that binds to the receiving cells. It is still unclear how Hedgehog can be activated and released to reach these faraway cells. One hypothesis is that an enzyme comes to the clusters and frees the proteins by cutting both of Hedgehog’s fatty anchors. Thanks to how the palmitate tethers Hedgehog to the cell, the protein is positioned in such a way that when the enzyme makes its snip, the binding site on the neighboring Hedgehog gets exposed: this protein is activated and, when also cut by the enzyme, released. Here, Schürmann et al. create an array of mutant Hedgehog proteins – for example some without palmitate, some with palmitate that cannot be removed by the enzyme – and study how they affect the development of the wing’s pattern in the fruit fly. Coupled with the imaging of the clusters, these experiments support the hypothesis that the palmitate anchor is necessary so that Hedgehog proteins can be turned on before diffusing away. The Hedgehog family of proteins is also present in humans, where it presides over the development of the embryo but is also involved in cancer. Understanding how Hedgehog works in the fruit fly could lead to new discoveries in humans too.

Nature Communications 6: Article number: 7520 (2015); Published: 3 July 2015; Updated: 21 January 2016. The authors inadvertently omitted Sumit Jaiswal, who was originally included in the Acknowledgements section of this Article and contributed to the cloning of transgenic constructs and the balancing of transgenic flies, from the author list.

In Drosophila, the development of the midline cells of the embryonic ventral nerve cord depends on the function of the bHLH-PAS transcription factor Single-minded (Sim). The expression domain of sim, however, is also found anterior and posterior to the developing ventral cord throughout the germ band. Indeed, mutations in sim were identified based on their characteristic cuticle phenotype. Eight abdominal segments (A1–A8) can be easily seen in the larval cuticle, while three more can be identified during embryogenesis. Cells located in A8–A10 give rise to the formation of the genital imaginal discs, and a highly modified A11 segment gives rise to the anal pads that flank the anus. sim is expressed in all these segments and is required for the formation of both the anal pads and the genital imaginal discs. A new temperature-sensitive sim allele allowed an assessment of possible postembryonic function(s) of sim. Reduction of sim function below a 50% threshold leads to sterile flies with marked behavioral deficits. Most mutant sim flies were only able to walk in circles. Further analyses indicated that this phenotype is likely due to defects in the brain central complex. This brain region, which has previously been implicated in the control of walking behavior, expresses high levels of nuclear Sim protein in three clusters of neurons in each central brain hemisphere. Additional Sim localization in the medullary and laminar neurons of the optic lobes may correlate with the presence of ectopic axon bundles observed in the optic lobes of sim mutant flies.

Dominant mutations in five tRNA synthetases cause Charcot–Marie–Tooth (CMT) neuropathy, suggesting that altered aminoacylation function underlies the disease. However, previous studies showed that loss of aminoacylation activity is not required to cause CMT. Here we present a Drosophila model for CMT with mutations in glycyl-tRNA synthetase (GARS). Expression of three CMT-mutant GARS proteins induces defects in motor performance and motor and sensory neuron morphology, and shortens lifespan. Mutant GARS proteins display normal subcellular localization but markedly reduce global protein synthesis in motor and sensory neurons, or when ubiquitously expressed in adults, as revealed by FUNCAT and BONCAT. Translational slowdown is not attributable to altered tRNA[superscript Gly] aminoacylation, and cannot be rescued by Drosophila Gars overexpression, indicating a gain-of-toxic-function mechanism. Expression of CMT-mutant tyrosyl-tRNA synthetase also impairs translation, suggesting a common pathogenic mechanism. Finally, genetic reduction of translation is sufficient to induce CMT-like phenotypes, indicating a causal contribution of translational slowdown to CMT., National Institutes of Health (U.S.) (Grant GM17151)

Mutanlallemand (mtl) and Belly Spot and Deafness (bsd) are two new spontaneous alleles of the Lmx1a gene in mice. Homozygous mutants show head tossing and circling behaviour, indicative of vestibular defects, and they have short tails and white belly patches of variable size. The analysis of auditory brainstem responses (ABR) showed that mtl and bsd homozygotes are deaf, whereas heterozygous and wildtype littermates have normal hearing. Paint-filled inner ears at E16.5 revealed that mtl and bsd homozygotes lack endolymphatic ducts and semicircular canals and have short cochlear ducts. These new alleles show similarities with dreher (Lmx1a) mutants. Complementation tests between mtl and dreher and between mtl and bsd suggest that mtl and bsd are new mutant alleles of the Lmx1a gene. To determine the Lmx1a mutation in mtl and bsd mutant mice we performed PCR followed by sequencing of genomic DNA and cDNA. The mtl mutation is a single point mutation in the 3' splice site of exon 4 leading to an exon extension and the activation of a cryptic splice site 44 base pairs downstream, whereas the bsd mutation is a genomic deletion that includes exon 3. Both mutations lead to a truncated LMX1A protein affecting the homeodomain (mtl) or LIM2-domain (bsd), which is critical for LMX1A protein function. Moreover, the levels of Lmx1a transcript in mtl and bsd mutants are significantly down-regulated. Hmx2/3 and Pax2 expression are also down-regulated in mtl and bsd mutants, suggesting a role of Lmx1a upstream of these transcription factors in early inner ear morphogenesis. We have found that these mutants develop sensory patches although they are misshapen. The characterization of these two new Lmx1a alleles highlights the critical role of this gene in the development of the cochlea and vestibular system.

MicroRNAs (miRNAs) are small noncoding RNAs able to regulate a broad range of protein-coding genes involved in many biological processes. miR-96 is a sensory organ-specific miRNA expressed in the mammalian cochlea during development. Mutations in miR-96 cause nonsyndromic progressive hearing loss in humans and mice. The mouse mutant diminuendo has a single base change in the seed region of the Mir96 gene leading to widespread changes in the expression of many genes. We have used this mutant to explore the role of miR-96 in the maturation of the auditory organ. We found that the physiological development of mutant sensory hair cells is arrested at around the day of birth, before their biophysical differentiation into inner and outer hair cells. Moreover, maturation of the hair cell stereocilia bundle and remodelling of auditory nerve connections within the cochlea fail to occur in miR-96 mutants. We conclude that miR-96 regulates the progression of the physiological and morphological differentiation of cochlear hair cells and, as such, coordinates one of the most distinctive functional refinements of the mammalian auditory system.

Aortic arch artery patterning defects account for approximately 20% of congenital cardiovascular malformations and are observed frequently in velocardiofacial syndrome (VCFS). In the current study, we screened for chromosome rearrangements in patients suspected of VCFS, but who lacked a 22q11 deletion or TBX1 mutation. One individual displayed hemizygous CHD7, which encodes a chromodomain protein. CHD7 haploinsufficiency is the major cause of coloboma, heart defect, atresia choanae, retarded growth and development, genital hypoplasia, and ear anomalies/deafness (CHARGE) syndrome, but this patient lacked the major diagnostic features of coloboma and choanal atresia. Because a subset of CHARGE cases also display 22q11 deletions, we explored the embryological relationship between CHARGE and VCSF using mouse models. The hallmark of Tbx1 haploinsufficiency is hypo/aplasia of the fourth pharyngeal arch artery (PAA) at E10.5. Identical malformations were observed in Chd7 heterozygotes, with resulting aortic arch interruption at later stages. Other than Tbx1, Chd7 is the only gene reported to affect fourth PAA development by haploinsufficiency. Moreover, Tbx1+/–;Chd7+/– double heterozygotes demonstrated a synergistic interaction during fourth PAA, thymus, and ear morphogenesis. We could not rescue PAA morphogenesis by restoring neural crest Chd7 expression. Rather, biallelic expression of Chd7 and Tbx1 in the pharyngeal ectoderm was required for normal PAA development.